Coronavirus Pseudotypes for All Circulating Human Coronaviruses for Quantification of Cross-Neutralizing Antibody Responses

Viruses. 2021 Aug 10;13(8):1579. doi: 10.3390/v13081579.


The novel coronavirus SARS-CoV-2 is the seventh identified human coronavirus. Understanding the extent of pre-existing immunity induced by seropositivity to endemic seasonal coronaviruses and the impact of cross-reactivity on COVID-19 disease progression remains a key research question in immunity to SARS-CoV-2 and the immunopathology of COVID-2019 disease. This paper describes a panel of lentiviral pseudotypes bearing the spike (S) proteins for each of the seven human coronaviruses (HCoVs), generated under similar conditions optimized for high titre production allowing a high-throughput investigation of antibody neutralization breadth. Optimal production conditions and most readily available permissive target cell lines were determined for spike-mediated entry by each HCoV pseudotype: SARS-CoV-1, SARS-CoV-2 and HCoV-NL63 best transduced HEK293T/17 cells transfected with ACE2 and TMPRSS2, HCoV-229E and MERS-CoV preferentially entered HUH7 cells, and CHO cells were most permissive for the seasonal betacoronavirus HCoV-HKU1. Entry of ACE2 using pseudotypes was enhanced by ACE2 and TMPRSS2 expression in target cells, whilst TMPRSS2 transfection rendered HEK293T/17 cells permissive for HCoV-HKU1 and HCoV-OC43 entry. Additionally, pseudotype viruses were produced bearing additional coronavirus surface proteins, including the SARS-CoV-2 Envelope (E) and Membrane (M) proteins and HCoV-OC43/HCoV-HKU1 Haemagglutinin-Esterase (HE) proteins. This panel of lentiviral pseudotypes provides a safe, rapidly quantifiable and high-throughput tool for serological comparison of pan-coronavirus neutralizing responses; this can be used to elucidate antibody dynamics against individual coronaviruses and the effects of antibody cross-reactivity on clinical outcome following natural infection or vaccination.

Keywords: COVID-19; SARS-CoV-2; coronavirus; neutralization; pseudotyped virus.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Viral / blood
  • Antibodies, Viral / immunology*
  • Broadly Neutralizing Antibodies / blood
  • Broadly Neutralizing Antibodies / immunology*
  • COVID-19 / immunology*
  • Cell Line
  • Coronavirus / immunology*
  • Coronavirus 229E, Human / immunology
  • Coronavirus 229E, Human / physiology
  • Coronavirus NL63, Human / immunology
  • Coronavirus NL63, Human / physiology
  • Coronavirus OC43, Human / immunology
  • Coronavirus OC43, Human / physiology
  • Cross Reactions
  • Humans
  • Lentivirus / genetics
  • Middle East Respiratory Syndrome Coronavirus / immunology
  • Middle East Respiratory Syndrome Coronavirus / physiology
  • Neutralization Tests
  • Plasmids
  • SARS-CoV-2 / immunology*
  • SARS-CoV-2 / physiology
  • Spike Glycoprotein, Coronavirus / immunology*
  • Transfection
  • Virus Internalization


  • Antibodies, Viral
  • Broadly Neutralizing Antibodies
  • Spike Glycoprotein, Coronavirus
  • spike protein, SARS-CoV-2